Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by significant deficits in reciprocal social interactions, impaired communication and restricted, repetitive behaviors, with a prevalence of 1 in 68 children in the US. Early intervention programs have been shown to be effective for improving IQ, language, and social skills for toddlers as young as 18 months of age. Diagnosis of ASD is currently based solely on clinical and parental observations, as a laboratory-based diagnostic test for ASD based on robust biomarkers does not exist currently. Our team previously demonstrated that lymphoblastoid cell lines (LCLs) from individuals with ASD generate reduced metabolic activity when the amino acid tryptophan is the only available energy source, relative to typically developing (TD) controls. In a recent evaluation of 50 ASD and 50 TD cell lines from individuals aged 1 to 10, a classifier utilizing a Bootstrap aggregation approach based upon tryptophan and an expanded series of ions was able to properly classify individual cell lines with both a sensitivity and specificity of 92% relative to the clinical diagnosis. The classifier showed better performance in the younger age groups, particularly in individuals aged 1 to 3, which is also the ideal target range for a screening test for ASD. The development of a laboratory biomarker test based on these patented methods will potentially serve as a second tier objective test following initial behavioral observations have identified a child at risk of developing ASD, thereby providing for confirmation of the diagnosis and earlier treatment. Potentially, such a test will also (1) lead to a better understanding of the pathogenic mechanisms underlying this condition, (2) assist in the identification of biochemical signatures delineating ASD sub-groups, and (3) highlight novel targets for improved and individualized treatment approaches. Widespread use of the existing test methods, however, is currently limited either by the cost and time associated with generating immortalized cell lines for each sample or by the size of the fresh blood sample that can be acquired from children, which is necessary to obtain sufficient numbers of live cells for the metabolic profiling assay. Therefore, the objective of the proposed effort is to develop methods and technologies to perform metabolic profiling on immune cells from small volumes of fresh blood to enable a viable screening test for ASD. Efforts will first be focused on improving the sensitivity of the current live-cell metabolic profiling techniques. Miniaturization of the assay platform will then be implemented to reduce the number of cells needed for each test condition, thereby limiting the necessary blood sample volume. Blood samples from a minimum of 100 subjects diagnosed with ASD and 100 TD controls will undergo metabolic profiling in a blinded fashion according to the methods and protocols developed. The performance of multiple classifiers will then be evaluated to determine the ability of the techniques to identify individuals diagnosed with ASD. The successful completion of this effort will lead directly to the Phase 2 investigation, which will involve testing an expanded cohort, including other neurodevelopmental disorders, and finalizing the design of a commercially viable testing service, platform, or kit. Commercialization of the technology proposed will ultimately provide for a screening test to identify toddlers at increased risk for ASD, thereby providing for earlier diagnosis and improved behavioral outcomes.